ABSTRACT Hydrogen peroxide (H2O2) is a versatile oxidant that mediates numerous biological functions within every major organ system. An emerging molecular pathway by which H2O2 accomplishes functional diversity is through the specific modification of protein cysteine residues to form S-sulfenylcysteine. This post-translational modification, S-sulfenylation, regulates protein activity and localization. Despite considerable advances with individual proteins, the biological chemistry, the dependency on specific H2O2-generating NADPH oxidases (Nox), and the structural elements that govern the modification of specific cysteine residues in vivo are vastly unknown. To provide insights into these fundamental biological questions, sensitive, validated, and quantitative chemical proteomic approaches are needed, but remain at an early stage of development. To this end, during the last funding period we developed and implemented a novel chemical proteomic approach. This new method has achieved specific, efficient, complementary and selective identification of S-sulfenylated cysteine residues in living cells. Currently, implementation of our chemoproteomic method has precisely pinpointed the site of S-sulfenylation in 1,105 peptides on 778 proteins in cultured mammalian cells. These proteins constitute the largest dataset of S-sulfenylated proteins reported to date. In this renewal application, we propose to use and expand our state-of-art chemical proteomic platform towards the three major objectives of: (1) defining the molecular determinants that govern the selection of specific cysteine resides and proteins for S-sulfenylation, (2) elucidating the functional networks and signaling pathways that are influenced by S-sulfenylation, and (3) identifying the enzyme system(s) that control protein desulfenylation. By uncovering the endogenous S- sulfenylome proteomics of mouse liver, brain, lung, and heart and applying multiple analytical and computational tools, the biochemical and structural properties that govern the specificity of S-sulfenylation in vivo will be defined. Biological functional and pathway analyses, in conjunction with quantitative stoichiometric assessment of S-sulfenylomes derived from Nox knockout and transgenic mice, will test H2O2-specific functional regulation in signaling cascades within and across the four different organs. Simultaneous acquisition of the endogenous site-specific, reactive cysteinome and phosphoproteome will enable comprehensive and global evaluation of complementation and coordination. Enzyme system(s) that regulate desulfenylation will be identified using CRISPR sequence-specific repression or activation of likely candidates. Overall, the comprehensive large-scale study of protein structures and functional pathways will significantly improve our appreciation of S-sulfenylation in H2O2-mediated biology. The molecular components of these pathways may, in turn, represent new biomarkers and drug targets in the rapidly growing fields of ?redox biology and medicine?. The research tools and methods advanced in this proposal should also provide of general value for characterizing redox networks in a range of physiological and disease processes.